Bone marrow failure in children

What every physician needs to know about bone marrow failure in children:

Bone marrow failure (BMF) in children is usually acquired, but inherited causes of pancytopenia (hemoglobin [Hb] less than 10g/dL, platelets less than 50K/uL, absolute neutrophil count [ANC] less than 1500/uL), such as Fanconi anemia (FA), dyskeratosis congenita (DC), Shwachman-Diamond syndrome (SDS), and congenital amegakaryocytic thrombocytopenia (CAMT) must be excluded. Depending on presentation, single lineage failures of red cells (Diamond-Blackfan anemia [DBA]), granulocytes (severe congenital neutropenia [SCN]) and cyclic neutropenia (CN), or platelets (thrombocytopenia with absent radii [TAR]) and CAMT (which can present with thrombocytopenia alone) should be considered.

The diagnostic approach to a patient who presents with pancytopenia relies on a detailed history including infections, medications/toxins, and family history, as well as careful physical examination, and laboratory tests including bone marrow aspiration and biopsy. Figure 1 shows an overview flow diagram of the diagnostic approach. The detailed work-up for a new patient is complex and should be undertaken by, or if that is not possible, in collaboration with, a pediatric hematologist or center expert in the care of these patients. Patients fulfilling the criteria of pancytopenia or those with depression of two lineages usually need further work-up that includes a bone marrow aspirate and biopsy.

What features of the presentation will guide me toward possible causes and next treatment steps:

Acquired aplastic anemia (AA) accounts for approximately 70% of BMF in children, while 30% is due to inherited causes. AA in children has a peak incidence in the teenage years and is defined as pancytopenia with a hypocellular bone marrow, without fibrosis, infiltration or evidence of myelodysplasia.

The diagnosis depends on the presence of at least two of the following:

Hb less than 10g/dL

Platelets less than 50K/µL

ANC less than 1.5K/µL

Bone marrow (BM) cellularity is 25 to 50% and reticulocyte count less than 40K/µL. In severe AA, the BM cellularity is less than 25%, with reticulocytes less than 20K/µL, and ANC less than 0.5K/µL, while in very severe AA, the ANC is less than 0.2K/µL.

Inherited BMF may present at birth or later during childhood as single lineage depression or as pancytopenia, and can be associated with congenital anomalies, failure to thrive with poor growth, and in some conditions an increased susceptibility to malignancy, in particular leukemia and myelodysplasia, although several other cancers are associated with specific BMF disorders.

When do you need to get more aggressive tests:

Once the diagnosis of aplastic anemia is made, aggressive work-up to establish the cause is urgently indicated, including a bone marrow aspirate and biopsy with cytogenetics and fluorescence in situ hybridization (FISH) for myelodysplasia, investigation for infectious etiologies, and for inherited bone marrow failure syndromes.

What imaging studies (if any) will be helpful?

Imaging studies will depend on clinical presentation and suspected inherited BMF disorder. A chest X-ray is indicated to exclude infection and as a baseline for future comparison. For example, ultrasound of the abdomen is required in DBA and FA to exclude renal and hepatic abnormalities, but the finding of lymphadenopathy or hepatosplenomegaly should raise the question of possible hematologic malignancy.

Echocardiogram may be required if congenital heart disease is suspected clinically.

Radiography of hands and forearms can be indicated in DBA, FA and TAR. A skeletal survey or radiography of long bones and ribs can be useful in the diagnosis of metaphyseal dysostosis, a feature of SDS, as can pancreatic ultrasound for fatty replacement of the pancreas.

What therapies should you initiate immediately and under what circumstances – even if root cause is unidentified?

Blood (packed red blood cell [PRBC]) or platelet transfusion may be urgently required if the patient is severely anemic or bleeding due to thrombocytopenia. Principles are:

Tests that are affected by PRBC transfusion such as red cell adenosine deaminase (ADA) should be obtained before transfusion

Pre-transfusion boluses of fluid should be avoided because of the risk of precipitating cardiac failure on transfusion of red cells

- Severely anemic children require careful monitoring of transfusion, which is given slowly in small aliquots, to avoid this complication.

PRBC and platelets should be leukocyte depleted and also obtained from cytomegalovirus (CVM)-negative donors until CMV status in established

- Since hematopoietic stem cell transplant is a possible treatment option, it is important not to transfuse CMV-negative patients with CMV-positive blood products.

Platelet transfusion

- Source should be pheresis rather than single unit donations, to avoid the additional exposure to human leukocyte antigens (HLAs) or non-HLAs that occur with several single unit donations required for one platelet transfusion. Multiple transfusions are more likely to induce alloantibody formation and refractoriness to platelet transfusions.

PRBC are transfused if the patient is symptomatic (Hb usually less than 6 to 7g/dL)

- Platelet transfusions are usually recommended if the count is less than 10K/uL or less than 20K/ul in the presence of fever. Some patients have very little bleeding even with low counts, and in some centers, platelets are transfused with bleeding such as intraoral purpuric lesions.

Neutropenic patients with fever will almost always require admission to hospital for blood culture and intravenous antibiotics until bacterial sepsis has been ruled out.

What other therapies are helpful for reducing complications?

Pancytopenia

Symptomatic anemic and thrombocytopenic patients require regular transfusion support until the diagnosis can be established and definitive treatment is begun. As noted above in the section on immediate therapy, precautions should be taken to avoid allosensitization (leukocyte depleted transfusions given only when clinically indicated). Neutropenic patients are susceptible to bacterial and fungal infections. In children prophylactic antibiotic are not usually required, but if recurrent infections occur, a prophylactic antibiotic such as bactrim may be helpful. Febrile neutropenic patients who do not defervesce require the early consideration for introduction of anti-fungal therapy.

Patients with severe acquired aplastic anemia should be HLA typed along with siblings and parents, to determine whether an HLA-matched sibling donor can be identified. If so, a transplant can be arranged as soon as inherited causes of BMF have been excluded, since this therapy offers the best prognosis and an opportunity for cure, with long term survival (approximately 90%).

Patients who do not have an HLA-matched sibling should be screened for an unrelated matched donor; potential donors are screened against the recipient by molecular high resolution typing to identify the best match. While this search proceeds patients are admitted for treatment with horse antithymocyte globulin (ATG) and cyclosporine, the levels of which have to be carefully monitored; steroids are also given as part of this protocol and can alleviate serum sickness, a complication of ATG treatment; approximately 70% of patients will partially or completely remit, with longer term survival around 80 to 90%.

Relapses can occur, and at that stage the usual approach is a matched unrelated transplant if a donor has been identified. ATG/cyclosporine patients require long term follow-up since patients are at risk of developing clonal BM abnormalities including myelodysplasia and acute leukemia; they are never cured of the AA and subtle evidence of stress erythropoiesis is usually present.

Fanconi anemia usually presents with macrocytosis (even without anemia) and thrombocytopenia during the first decade, and then trilineage BMF subsequently ensues. The goal of management is to avoid transfusions, treat infections early and aggressively, coordinate care for other associated physical anomalies and endocrine abnormalities, and provide cancer and MDS/leukemia surveillance.

Management requires judicious use of transfusions, and stimulation of hematopoiesis with an androgen is frequently tried as a first measure if transfusion dependence occurs. Androgens are generally avoided if an unaffected HLA-identical sibling donor is available. Granulocyte colony-stimulating factor (G-CSF) may be helpful for severe neutropenia and is generally recommended for consistent ANC less than 500/uL. Hematopoietic stem cell transplantation (HSCT) is the desired option if a matched unaffected sibling is available, and if not, the options for unrelated matched HCT should be explored.

Treatment of dyskeratosis congenita follows the same lines. Shwachman-Diamond patients are often chronically or intermittently neutropenic during childhood, and this can progress to pancytopenia. Congenital amegakaryocytic thrombocytopenia is very rare, and would usually (but not always) present with thrombocytopenia in infancy and can progress to pancytopenia, since the thrombopoietin receptor (myeloproliferative leukemia virus oncogene [MPL]) is expressed on hematopoietic stem cells. Most patients with inherited BMF syndromes will have macrocytosis, even if they are not anemic or pancytopenic. It is critical to review the smear of patients to look for macrocytes and to be aware of the MCV of previous CBCs.

Because of their DNA repair defect, FA patients are at greatly increased risk of developing leukemia as well as gastrointestinal (GI), genitourinary (GU) and other tumors, in particular hepatic. The telomere loss in DC patients is also associated with an increased risk of malignancy, particularly squamous cell tumors of the oropharynx, GI tract and skin, but other tumors as well. Patients with SDS are susceptible to AML and MDS.

All inherited BMF patients require long term follow-up, including annual BM aspirates/biopsies to monitor for the development of clonal abnormalities. Long term care in patients with other congenital abnormalities frequently requires multidisciplinary help from appropriate colleagues with expertise in different disciplines.

Single lineage failure

DBA: The challenge in an infant with pure red cell aplasia (no retics, no or few erythroblasts in BM) is to distinguish DBA from transient erythroblastopenia (TEC). Younger age at presentation (less than 1 year), other typical congenital abnormalities, high MCV and or fetal hemoglobin (HbF), and high erythrocyte adenosine deaminase (eADA) done before transfusion can be helpful in the diagnosis of DBA. Management hinges on transfusion, steroids (not started until 1 year of age), and failure to respond requires chronic transfusion and raises possibility of HSCT, the only known cure.

Severe congenital neutropenia (SCN) and cyclic neutropenia (CN): These patients usually have severe neutropenia from infancy and have recurrent sinopulmonary and skin infections, in contrast to the more common chronic benign neutropenia of childhood, which usually presents at around 1 year of age and is not associated with frequent infections, although it can persist for years (median recovery approximately 2 years).

SCN patients usually respond very well to G-CSF, which is required long term. The improvement in survival has brought out the increased risk of leukemia to which SCN patients are susceptible. CN patients usually follow a much milder course, often present later, and frequently do not require therapy. Some patients may require G-CSF support, however, although the doses required are usually substantially lower than those used in patients with SCN.

What should you tell the patient and the family about prognosis?

Prognosis very much depends on the precise diagnosis and needs to be discussed on several occasions with the patient and family as the investigation proceeds and the diagnosis becomes clarified. Initially, if it appears likely that severe aplastic anemia or an inherited bone marrow failure syndrome is most likely, a conversation along the lines of a "Day One Talk" in the context of a serious illness such as cancer is indicated, emphasizing the importance of accurate diagnosis, that good treatment is available with the goal of cure in some contexts, that acquired causes are infrequently identified, and that parents are not to blame for what has happened.

At a later conversation, once the diagnosis has been confirmed, it is more appropriate to discuss prognosis as it relates to the specific treatment options indicated for that disease. What the treatment involves, side effects of medications, and risks of complications during treatment require discussion. Since bone marrow transplantation is a treatment option for many of these conditions, an early hematopoietic stem cell transplant (HSCT) consult is desirable.

Particularly if unsuspected, the diagnosis of bone marrow failure is an enormous event for both patient and family. The management of these diseases with frequent hospital visits will be very disruptive, and being forewarned of the difficulties may be helpful.

“What if” scenarios.

A common pitfall is a child who presents with thrombocytopenia and is misdiagnosed as immune thrombocytopenic purpura (ITP). Fanconi anemia (FA) can typically present during the first decade of life as thrombocytopenia, and only with time does more general bone marrow failure and pancytopenia manifest. Dyskeratosis congenita (DC), Shwachman-Diamond syndrome (neutropenia is a more common presenting hematologic feature) and congenital amegakaryocytic thrombocytopenia (thrombocytopenia from an early age) need to be considered as well. A careful history and examination for short stature, skin lesions such as pigmented or depigmented areas (FA), facial appearance, a lacy reticulate rash on the neck, leukoplakia, or dystrophic nails (DC).

Another common scenario is a patient diagnosed with acquired aplastic anemia that has received medical treatment with ATG/cyclosporine and in fact has an inherited BMF syndrome.

Careful screening of HSCT donors in inherited BMF syndromes is critical, as transplantation from a sibling unsuspected of having the disease but who is, in fact affected, can be disastrous. Genetic diagnosis, if available, is most helpful. If a genetic lesion has not been identified, then a careful search must be made for clinical or laboratory features of the disease, since expressivity of the phenotype can vary widely within families.

Pathophysiology

Acquired aplastic anemia

Acquired aplastic anemia can be secondary to infections such as hepatitis and Epstein-Barr virus (EBV), to toxins such as organophosphates and benzene, or to drugs such as chloramphenicol, but in most cases the disease is idiopathic. There is evidence to support the notion that idiopathic AA is due to an autoimmune attack on hematopoietic stem cells (HSC) by T lymphocytes, but a stem cell antigen to which this attack is directed has never been defined. It is possible that somatic or inherited mutations in genes important for HSC function or for immune regulation underlie this disease.

Fanconi anemia

FA is inherited in an autosomal recessive or X-linked pattern. Mutations in any of 15 genes that play a role in the repair of DNA interstrand cross-links (ICL) can manifest as FA, resulting in congenital malformations, progressive bone marrow failure, and increased susceptibility to cancer. The defect in DNA repair results in sensitivity to DNA cross-linking agents such as cisplatin, diepoxybutane and mitomycin C. The FA pathway comprises eight core complex FANC proteins that, together with FA-associated proteins, lead to mono-ubiquitination of Fanconi anemia group D2 protein (FANCD2) and Fanconi anemia, complementation group I (FANCI). These two proteins are recruited to sites of ICL where they are de-ubiquitinated before playing a role, along with other FANC proteins in several pathways of ICL repair.

Shwachman-Diamond syndrome

SDS is due to autosomal recessively inherited mutations in the Shwachman Bodian Diamond (SBDS) gene (90% of cases). Although the pathogenesis of SDS is not fully understood, it can be classified as a ribosomopathy, along with Diamond-Blackfan anemia, dyskeratosis congenita, cartilage-hair hypoplasia and Treacher Collins syndrome. The SBDS protein plays a role in maturation of the large 60S ribosomal subunit, but is also a multifunctional protein that has functions in mitotic spindle stabilization, actin polymerization, vacuolar pH regulation and DNA metabolism. The precise role of defects in these pathways in the bone marrow failure (in particular neutropenia), exocrine pancreatic deficiency with malabsorption, skeletal defects, and cancer susceptibility is not understood.

Dyskeratosis congenita

DC is due to a defect in telomere maintenance. Telomeres are complex structures at the ends of chromosomes, which shorten with each cell division and are maintained by the telomerase enzyme, a ribonucleoprotein complex consisting of a catalytic reverse transcriptase component telomerase reverse transcriptase (TERT), its ribonucleic acid (RNA) component telomerase RNA component (TERC), and four other H/ACA RNA associated proteins, dyskerin, NOP10, NHP2, and GAR1.

That telomerase is defective in DC came from the discovery that the X-linked form of DC is due to mutations in the gene for dyskerin, DKC1. Dyskerin, NOP10 and NHP2 also bind to H/ACA small nucleolar (sno) RNAs, and play a role in pseudouridylation of nascent ribosomal RNA; whether defects in this process contribute to the DC phenotype is uncertain. Autosomal recessive forms of DC are due to mutations in TERT, TERC, NOP10 and NHP2, and the TERT and TERC mutations are associated with classic DC (dyskeratotic nails, rash and leukoplakia), with later development of bone marrow failure.

Heterozygous mutations in TERT and TERC or NOP10/NHP2 homozygous mutations can result in milder forms of DC with later complications such as cancer, myelodysplastic syndrome/acute myeloid leukemia, pulmonary or liver fibrosis. Telomeres are protected by a shelterin complex of six proteins, one of which is TIN2; heterozygous mutations in TINF2, the gene encoding TIN2, cause severe forms of DC. Since telomerase expression is confined to a few cell types including hematopoietic stem cells, it is possible that dysfunctional telomeres lead to p53 stabilization and cell cycle arrest or cell death, and that cells that escape this process may be potentially malignant.

Congenital amegakaryocytic thrombocytopenia

Congenital amegakaryocytic thrombocytopenia is due to homozygous or compound heterozygous mutations in the receptor for thrombopoietin, MPL. Thrombocytopenia is usually present from birth and trilineage aplasia at a young age, because the receptor is present on HSC.

Diamond-Blackfan anemia

Diamond-Blackfan anemia was the first human disease associated with a dominantly inherited mutation in a ribosomal protein (RP) gene, RPS19, in 25% cases. Eleven other RP genes have now been shown to be mutated in DBA, the commonest are RPL5, RPL11, and RPS26. Altogether, known RP mutations account for approximately 50% of cases. The mutations usually result in haploinsufficiency of the protein.

Analysis of ribosome biogenesis in DBA patients and model systems shows that the different RP gene mutations result in a block in ribosome biogenesis. Recent data from several laboratories suggest that ribosomal stress, which is a consequence of the defect in ribosome biogenesis, leads to stabilization of p53 and cell death by apoptosis. Why erythroid progenitors are preferentially affected is uncertain, but may relate to the rapid proliferation and high demand for RNA synthesis in the cells during early stages of erythropoiesis, particularly during late fetal development.

What other clinical manifestations may help me to diagnose bone marrow failure?

What other additional laboratory studies may be ordered?

Additional studies:

Diepoxybutane (DEB) test for FA

Telomere length measurement (DC)

Gene sequencing if inherited BMF syndrome suspected

- FA gene sequencing is not indicated except under special circumstances such as prenatal genetic diagnosis of a sibling. SBDS and MPL gene sequencing if clinically indicated. If telomeres are short or DC is suspected, sequencing of the six DC genes may be indicated. DBA strongly suspected: Reasonable to sequence seven genes, RPS19, RPL5, RPL11, RPL35A, RPS26, RPS10, and RPS24.

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